TFAM-AGER通路介导结肠癌细胞免疫原性死亡的机制研究

Immunogenic cell death (ICD) is a type of cell death that is accompanied by the release of damage-associated molecular patterns (DAMPs) and results in a dead-cell antigen-specific immune response. Here, we report that spautin-1, an inhibitor of ubiquitin-specific peptidases, triggers immunogenic cancer cell death in vitro and in vivo. The anticancer activity of spautin-1 is independent of autophagy inhibition, but it depends on the intrinsic mitochondrial apoptosis pathway. Spautin-1 causes mitochondrial oxidative injury, which results in JUN transcription factor activation in a JNK-dependent manner. Mechanistically, activation of JUN by spautin-1 leads to apoptosis by upregulation of pro-apoptotic BAD expression. Furthermore, the release of TFAM, a mitochondrial DAMP, by apoptotic cells may contribute to spautin-1-induced ICD via its action on the receptor AGER. It is discovered that cancer cells treated with spautin-1 in vitro were able to elicit an anticancer immune response when inoculated in vivo in the absence of any adjuvant. Also, this immunogenic effect of spautin-1-treated cancer cells was lost when TFAM or AGER was neutralized by specific antibodies. Altogether, our results suggest that spautin-1 may stimulate an apoptotic pathway that results in ICD, in a TFAM-dependent and AGER-dependent fashion. In this project, we will exploit molecular, cellular, and transgenic animal models to pursue the following aims. Aim 1. Define the role of BAD in the regulation of TFAM release during ICD. Aim 2. Define the role of AGER in the regulation of TFAM activity during ICD. Aim 3. Evaluate the anticancer activity of TFAM/AGER-mediated ICD in mouse models. To conclude, this project plans to fully utilize our prior work and provide a more in-depth understanding of TFAM-AGER pathway in ICD. Our hypothesis is novel and the results may lead to innovative therapeutics for the treatment of colon cancer.

以肿瘤细胞免疫原性死亡（ICD）为基础的肿瘤疫苗为肿瘤的预防和治疗开辟了新的方向。申请人的原创性研究表明小分子化合物Spautin-1能够诱导肿瘤细胞ICD，而凋亡细胞释放的线粒体转录因子A (TFAM) 作为一种ICD介质能更有效地将肿瘤抗原提呈给机体的免疫系统，由此产生针对肿瘤的免疫杀伤效应。在此研究基础之上，提出“活化TFAM-AGER通路是介导癌细胞ICD的关键”的科学假说。针对上述假说，本项目拟选用基因工程动物、结肠癌细胞和树突状细胞为主要研究材料，采用CRISPR/Cas9基因编辑和位点突变等研究手段，在整体、细胞和分子水平，进一步深入系统地探讨TFAM-AGER通路调控癌细胞ICD的分子机制、结构基础和信号转导，探索肿瘤疫苗防治结肠癌的路径。开展本项目有助于阐明ICD的关键通路，为肿瘤免疫的研究开辟新的方向，为寻找结肠癌的治疗手段提供新的策略。

肿瘤细胞受到外界刺激发生死亡的同时，由非免疫原性转变为免疫原性而介导机体产生抗肿瘤免疫应答的过程称为免疫原性细胞死亡（immunogenic cell death，ICD）。肿瘤细胞发生ICD的同时，会产生一系列的信号分子，此类物质被称为损伤相关分子模式(DAMP)。ICD过程中释放的DAMPs能够和DC细胞表面的模式识别受体PRRs结合，启动一系列的细胞学反应，最终激活先天和适应性免疫反应。本项目主要探索肿瘤细胞发生免疫原性死亡的理论基础、相关信号分子作用机制以及它们在介导肿瘤免疫识别与杀伤中的应用价值。主要取得了下列成果。.1）BAD介导肿瘤细胞TFAM释放的磷酸化修饰和铁代谢。本项目运用各种BAD磷酸化突变体、基因敲除技术和药物干预手段证明spautin-1通过调节PKA与AKT活性，影响BAD磷酸化和线粒体铁的聚集，从而促进肿瘤细胞氧化损伤和TFAM释放。.2）AGER介导树突状细胞TFAM活性的位点结合结构基础和信号转导分子通路。本项目运用各种AGER结构突变体、基因敲除技术和药物干预手段证明TFAM通过与AGER胞外可变区直接结合，影响下游信号通路的活化，从而促进树突状细胞成熟、移位和活化。.3）TFAM-AGER通路介导结肠癌肿瘤疫苗活性的药物和转基因动物研究。利用FPS-ZM1（新型的AGER小分子抑制剂）和AGER条件性敲除小鼠阐明不同细胞来源的AGER如何相互作用，调控TFAM介导的抗肿瘤免疫活性。并且证明了CDK1/2/5 和HSP90的抑制克服了 IFNG 介导的胰腺癌适应性免疫抵抗通过诱导免疫原性死亡。.4) 铁死亡的分子机制和免疫原性。我们的研究证明，线粒体DNA应激通过STING途径触发自噬依赖性铁死亡； 并阐明 TMEM164 和 EIF4E 是影响肿瘤免疫原性和自噬反应的新型铁死亡调节蛋白，AGER介导了铁死亡的免疫原性。.本项目圆满完成既定目标，在《GUT》, 《NATURE COMMUNICAITON》、《AUTOPHAGY》和《CANCER RESEARCH》等期刊发表论文8篇，培养研究生4名，在学术会议上交流10次，上述成果对于开发新型的抗肿瘤治疗手段提供重要实验室参考。